Cecal Microbiome and Metabolism Response to Campylobacter Exposure and Casein Supplementation

The effect of the treatment was evaluated following a well-established protocol for the C. jejuni cecal model, as outlined by Olson et al. and Feye et al. [16 (link),19 ]. Cecal contents were collected from six individual birds within aseptic chambers, then weighed and diluted at a ratio of 1:3000 by mixing 0.1 g of cecal content with 900 μL of ADS solution. This resuspended cecal material was further diluted by adding 1 mL of it to 299 mL of ADS for each respective cecum sample. A 36 mL aliquot of this diluted cecal content was subsequently transferred into each serum bottle, with or without the addition of 0.4 g of ground chicken feed (S1 Table). To create an appropriate environment, the cultures were covered with aluminum foil and placed in Advanced Anoxomat^™ III containers (Advanced Instruments, Norwood, MA, USA), where a microaerobic atmosphere was established, containing 5% O2, 10% CO2, and 85% N2. Incubation was carried out at 42°C with continuous agitation at 150 revolutions per minute (RPM) for 24 hours. This step served as a pre-adaptation to acclimate the naturally occurring microbiota in poultry ceca to the new environment, following the method described previously [18 (link)–20 (link)]. On the following day, the serum bottles were returned to the anaerobic chamber.
After 24 h of pre-adaption, the serum bottles were moved back into the anaerobic chamber where 4 ml of a freshly prepared 10⁶ cells/mL C. jejuni culture resuspended in ADS was added to all bottles except the non-inoculated treatment (NI) for a final concentration of 10⁵ CFU/mL. For specific treatments involving casein supplements, 0.1 g of the appropriate vitamin free-casein type (intact (Envigo®, Madison, WI, USA), enzyme hydrolyzed (MilliporeSigma, Burlington, MA, USA), or acid hydrolysate of casein (MilliporeSigma, Burlington, MA, USA) was added to the serum bottles. The study included five distinct treatments: non-inoculated (NI), inoculated with C. jejuni (IN), and three supplemented groups, intact casein (IC), enzyme hydrolysate (EH), acid hydrolysate (AH), that were all inoculated with C. jejuni. We opted not to include additional controls for supplemented groups without Campylobacter, instead using non-inoculated and inoculated treatments without casein supplementations for comparative analyses with the casein-enriched groups. Because Campylobacter naturally inhabits poultry cecal compartments [21 (link)], we confirmed the absence of culturable Campylobacter in the NI group and confirm the inocula, the 0-hour samples were plated on mCCDA [22 (link)]. At 0, 24, and 48 h post-inoculation, duplicate 1 mL samples were taken for microbiome sequencing and metabolomic analysis. The samples were flash-frozen in liquid nitrogen and stored at -80°C until processing. An overview of the procedure is described in Fig 1.